1. Field of the Invention
The basic unit of information transmitted from one part of the nervous system to another is a single action potential or nerve impulse. The xe2x80x9ctransmission linexe2x80x9d for these impulses is the axon, or nerve fiber. The electrical excitability of the nerve membrane has been shown to depend on the membrane""s voltage-sensitive ionic permeability system that allows it to use energy stored in ionic concentration gradients. Electrical activity of the nerve is triggered by a depolarization of the membrane, which opens channels through the membrane that are highly selective for sodium ions, which are then driven inward by the electrochemical gradient. Of the many ionic channels, the voltage-gated or voltage-sensitive sodium channel is one of the most studied. It is a transmembrane protein that is essential for the generation of action potentials in excitable cells.
The cDNAs for several Na+ channels have been cloned and sequenced. These studies have shown that the amino acid sequence of the Na+ channel has been conserved over a long evolutionary period. These studies have also revealed that the channel is a single polypeptide containing four internal repeats, or homologous domains (domains I-IV), having similar amino acid sequences. Each domain folds into six predicted transmembrane xcex1-helices or segments: five are hydrophobic segments and one is highly charged with many lysine and arginine residues. This highly charged segment is the fourth transmembrane segment in each domain (the S4 segment) and is likely to be involved in voltage-gating. The positively charged side chains on the S4 segment are likely to be paired with the negatively charged side chains on the other five segments such that membrane depolarization could shift the position of one helix relative to the other, thereby opening the channel. Accessory subunits may modify the function of the channel.
There is a significant therapeutic utility in recombinant materials derived from the DNA of the numerous sodium channels that have been discovered. For example, the recombinant protein can be used to screen for potential therapeutics that have the ability to inhibit the sodium channel of interest. In particular, it would be useful to inhibit selectively the function of sodium channels in nerve tissues responsible for transmitting pain and pressure signals without simultaneously affecting the function of sodium channels in other tissues such as muscle, heart and brain. Such selectivity would allow for the treatment of pain without causing side effects due to cardiac, central nervous system or neuromuscular complications. Therefore, it would be useful to have DNA sequences coding for sodium channels that are selectively expressed in peripheral sensory nerve tissue. Though cDNAs from rat skeletal muscle, heart and brain are known, identification and isolation of cDNA from peripheral sensory nerve tissue, such as dorsal root ganglia, has been hampered by the difficulty of working with such tissue.
This invention relates to a cloned xcex1-subunit of a voltage-gated tetrodotoxin-resistant sodium channel protein expressed in peripheral nerve tissue. This invention further relates to its production by recombinant technology and nucleic acid sequences encoding for this protein.
2. Summary of Related Art
An excellent review of sodium channels is presented in Catterall, TINS 16(12):500-506 (1993).
Purified Na+ channels have proven useful as therapeutic and diagnostic tools, Cherksey, U.S. Pat. No. 5,132,296.
The cDNAs for several Na+ channels have been cloned and sequenced. Numa, et al., Annals of the New York Academy of Sciences 479:338-355 (1986), describes cDNA from the electric organ of eel and two different ones from rat brain. Rogart, U.S. Pat. No. 5,380,836 describes cDNA from rat cardiac tissue. See also Rogart, Cribbs et al. Proc. Natl. Acad., Sci., 86:8170-8174 (1989). A peripheral nerve sodium channel, referred to as PN1, has been detected based on sodium current studies and hybridization to a highly conserved sodium channel probe by D""Arcangelo, et al., J. Cell Biol. 122:915-921 (1993). However, neither the DNA nor the protein were isolated and its complete nucleic acid and amino acid sequence remained unidentified. A partial amino acid sequence was presented at the 23rd Annual Meeting of the Society for Neuroscience, Nov. 7-12, 1993, Washington D.C., see Abstracts: Volume 19, Part 1: Abstract 121.7: xe2x80x9cNerve Growth Factor Treatment of PC12 Cells Induces the Expression of a Novel Sodium Channel Gene, Peripheral Nerve Type 1 (PN1)xe2x80x9d, by B. L. Moss, J. Toledo-Aral and G. Mandel.
Tetrodotoxin (xe2x80x9cTTXxe2x80x9d), a highly potent toxin from the puffer or Fugu fish, blocks the conduction of nerve impulses along axons and in excitable membranes of nerve fibers, which leads to respiratory paralysis. TTX also binds very tightly to the Na+ channel and blocks the flow of sodium ions. The positively charged group of the toxin interacts with a negatively charged carboxylate at the mouth of the channel on the extracellular side of the membrane, thus obstructing the conductance pathway.
Studies using TTX as a probe have shed much light on the mechanism and structure of Na+ channels. There are three Na+ channel subtypes, defined by the affinity for TTX which can be measured by the IC50 values: TTX-sensitive Na+ channels (IC50≈1 nM), TTX-insensitive Na+ channels (IC50=1-5 xcexcM), and TTX-resistant Na+ channels (IC50xe2x89xa7100 xcexcM)
TTX-insensitive action potentials were first studied in rat skeletal muscle. Redfern, et al., Acta Physiol. Scand. 82:70-78 (1971). Subsequently, these action potentials were described in other mammalian tissues, including newborn mammalian skeletal muscle, mammalian cardiac muscle, mouse dorsal root ganglion cells in vitro and in culture, cultured mammalian skeletal muscle and L6 cells. Rogart, Ann. Rev. Physiol. 43:711-725 (1980).
Dorsal root ganglia neurons possess both TTX-sensitive (IC50≅0.3 nM) and TTX-resistant (IC50≅100 xcexcM) sodium channel currents, as described in Roy, et al., J. Neurosci. 12:2104-2111 (1992).
TTX-resistant sodium currents have also been measured in rat nodose and petrosal ganglia, Ikeda, et al., J. Neurophysiol. 55:527-539 (1986) and Stea, et al., Neurosci. 47:727-736 (1992).
One aspect of the present invention is a purified and isolated DNA sequence encoding for a mammalian peripheral nerve sodium channel protein, in particular, the xcex1-subunit of this protein. A preferred embodiment of the invention is a purified and isolated DNA sequence encoding a mammalian peripheral nerve TTX-resistant sodium channel.
Further aspects of the invention include expression vectors comprising the DNA of the invention, host cells transformed or transfected by these vectors, specifically mammalian cells, and a cDNA library of these host cells.
Another aspect of the present invention is a recombinant polynucleotide comprising a nucleic acid sequence derived from the DNA sequence of this invention.
Still another aspect of the invention is the rat and human peripheral nerve TTX-resistant sodium channel protein encoded by the DNA of this invention.
Also forming part of this invention is an assay for inhibitors of the peripheral nerve TTX-resistant sodium channel protein comprising contacting a compound suspected of being an inhibitor with expressed sodium channel and measuring the activity of the sodium channel.
Further provided is a method of inhibiting the activity of the peripheral nerve TTX-resistant sodium channel comprising administering an effective amount of a compound having an IC50 of 10 xcexcM or less.